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    Analysis of Spa33 and its Role in T3SS Cytoplasmic Sorting Platform of Shigella

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    Issue Date
    2019-12-31
    Author
    Skaar, Ryan Richard
    Publisher
    University of Kansas
    Format
    85 pages
    Type
    Thesis
    Degree Level
    M.S.
    Discipline
    Pharmaceutical Chemistry
    Rights
    Copyright held by the author.
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    Abstract
    Enteric infections, particularly those leading to diarrhea, can profoundly disrupt intestinal function and have a major impact on global mortality and morbidity rates.1 Globally, there are 1.7 billion cases of childhood diarrheal disease every year, which results in the death of approximately 525,000 children under the age of 5.2 Four pathogens were identified to be the key contributors to childhood diarrheal cases. These pathogens include rotavirus, Cryptosporidium, Shigella, and ST-ETEC. While rotavirus is the most prominent contributor to diarrheal episodes, specifically in infants (0 – 11 months), Shigella’s influence grows and becomes the primary contributor to diarrheal episodes as the child reaches the toddler stage of development (24 – 59 months).3 Ingestion of Shigella bacterium results in the luminal infection referred to as shigellosis. Before symptoms appear, Shigella first crosses the colonic epithelium via M cells where it is engulfed by macrophages that then undergo pyroptosis. After release into the sub-mucosa, Shigella invades intestinal epithelial cells using its type III secretion system (T3SS). The T3SS or injectisome is essential for Shigella virulence. The injectisome consists of three major components: extracellular needle, basal body, and a cytoplasmic sorting platform. Effector secretion is triggered by host cell contact and controlled by the sorting platform (SP). Shigella’s SP is comprised of five essential virulence proteins (MxiG, MxiK, Spa33, MxiN, and Spa47), which are highly conserved across all Shigella species. Characterization of the cytoplasmic SP via cryo-electron tomography allowed us to generate a 3D model of the SP containing six pod-like structures (comprised most prominently of Spa33). Spa33 is then connected to MxiN spokes that link it to the central ATPase (Spa47) and to MxiK, which links it to the basal body. This model differs dramatically from the contiguous arrangement of C ring proteins seen within the distantly related Gram-negative flagellum. Deletion of spa33 results in complete loss of the SP. Spa33 consists minimally of a dimer of C-terminal Spa33 domains (Spa33C) and a full-length copy (Spa33FL). Spa33FL is unstable on its own, but is greatly stabilized by association with its alternatively translated Spa33C dimer. Here I provide support using various biophysical techniques in vivo and in vitro that the association between Spa33FL and Spa33C is essential to T3SS function, suggesting that both Spa33FL and Spa33C are necessary for SP assembly in Shigella.
    URI
    http://hdl.handle.net/1808/30201
    Collections
    • Pharmaceutical Chemistry Dissertations and Theses [141]
    • Theses [3906]

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    785-864-8983

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    1425 Jayhawk Blvd
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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